The aim of this work is the validation of an Euler–Lagrange modeling approach coupling a CFD-based compartment model (Eulerian approach) and a stochastic model based on a Continuous-Time Markov Chain ... [more ▼]

The aim of this work is the validation of an Euler–Lagrange modeling approach coupling a CFD-based compartment model (Eulerian approach) and a stochastic model based on a Continuous-Time Markov Chain (Lagrangian approach). The turbulent flow structure and the mixing process in a bioreactor stirred by an axial Mixel TT impeller is characterized by PIV and tracer experiments. Comparison between experimental and numerical data shows that the CFD-based compartment model is able to reproduce accurately the spatial heterogeneities inside the bioreactor. The trajectory of a small tracer particle which perfectly follows the fluid flow is measured by optical trajectography. It is then simulated by a stochastic model which is either based on an homogeneous or on an inhomogeneous Continuous Time Markov Chain (CTMC). Comparison of residence and circulation time distributions in three zones defined inside the bioreactor shows that the inhomogeneous CTMC model predicts with an excellent accuracy the particle trajectories inside the bioreactor. The modeling approach proposed here could be an useful tool to design scale-down bioreactors in order to reproduce at lab-scale the stress levels encountered in large-scale production bioreactors and to characterize and compare different bioreactor configurations. [less ▲]

The use of genetically encoded fluorescent reporters allows speeding up the initial optimization steps of microbial bioprocesses. These reporters can be used for determining the expression level of a ... [more ▼]

The use of genetically encoded fluorescent reporters allows speeding up the initial optimization steps of microbial bioprocesses. These reporters can be used for determining the expression level of a particular promoter, not only the synthesis of a specific protein but also the content of intracellular metabolites. The level of protein/metabolite is thus proportional to a fluorescence signal. By this way, mean expression profiles of protein/ metabolites can be determined non-invasively at a high-throughput rate, allowing the rapid identification of the best producers. Actually, different kinds of reporter systems are available, as well as specific cultivation devices allowing the on-line recording of the fluorescent signal. Cell-to-cell variability is another important phenomenon that can be integrated into the screening procedures for the selection of more efficient microbial cell factories. [less ▲]

Many mosquito (Diptera: Culicidae) species are zoonotic vectors responsible for numerous infectious diseases of medical and veterinary importance. Currently, changes in the vectors’ geographical distribution induced chiefly by anthropogenic factors are accompanied by emerging and reemerging infectious diseases in Europe and North America. Since the advent of synthetic insecticides during the Second World War, mosquitoes are the object of considerably expanded and deepened research. In an integrated pest management context, means of control are now mainly classified as: (1) environmental management and physical control, (2) chemical control, (3) genetic control, and (4) biological control by means of entomophagous predators and entomopathogenic microorganisms. In this context, these last have significant potential because of their ability to infect and kill their host with more or less targeted selectivity. This article proposes to emphasize biological control among other techniques in mosquito control, and to assess the potential and the opportunities offered by entomopathogenic bacteria, viruses and fungi. Finally, their use as biopesticides is discussed. [less ▲]

The goal of this research is to directly highlight the simultaneous occurrence of several phenotypes with distinct metabolic function among clonal population of E. coli., often used for recombinant ... [more ▼]

The goal of this research is to directly highlight the simultaneous occurrence of several phenotypes with distinct metabolic function among clonal population of E. coli., often used for recombinant protein and pDNA production in bioprocesses. This phenotypic heterogeneity, first due to the noise, is reinforced by environmental heterogeneities occurring at large scale during fed-batch processes. This phenotypic heterogeneity has been tracked according to GFP reporter strains (biosensors) that circulate in the bioreactor and encounter environmental heterogeneities. First, we have highlighted the simultaneous occurrence of several phenotypes with distinct metabolic functions. Indeed, a diversity of glucose uptake strategies has recently been noticed with the PtsG and MglABC transporters. Moreover, when E.coli encounters zones of glucose excess, acetate is produced through the overflow metabolism. Only the sub-population with high acs expression (acetate transporter) could consume this acetate. GFP reporter strains have thus been constructed for PtsG, MgIABC and acs genes. In addition of the stable GFP, two destabilized GFP variants for each gene have been used to obtain more instantaneous responses. The response of these biosensors have been followed by on-line flow cytometry. In the end, this experimental strategy for direct phenotyping at the single cell level will also be used to investigate the impact of metabolic engineering strategies on phenotypic heterogeneity, robustness and fitness of microbial population in industrial conditions. [less ▲]

At this time, many wastes are used or eliminated through processes that do not really consider their potential applications. Such wastes contain useful nutrients (nitrogen, phosphorus and potassium), the ... [more ▼]

At this time, many wastes are used or eliminated through processes that do not really consider their potential applications. Such wastes contain useful nutrients (nitrogen, phosphorus and potassium), the importance of which has been demonstrated in agriculture for many years. The composition of wastes is highly heterogeneous, which makes treatment techniques more difficult to apply on a large scale. Sewage sludge is usually used as a fertilizer in agriculture, in energy production or in the field of construction. The main use of manure is agriculture, although considerable amounts of nutrients are lost and cause pollution. Digestate is also used in agriculture, but other alternatives have been proposed. Ashes should also be highlighted, although they do not contain nitrogen, which is lost into the atmosphere during the combustion process. Finally, household and industrial wastes are resources that should be considered as well. Those different types of wastes could be recycled to produce environment-friendly fertilizers. Here, we propose to investigate these opportunities inside five European countries (Belgium, France, Germany, United Kingdom and The Netherlands) through five work packages with the BioRefine Project. [less ▲]

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on ... [more ▼]

The purpose of this work was the observation of the differences between the microbial communities living in the gut of the termite Reticulitermes flavipes fed on different diets. The termites were fed on poplar wood (original diet) and artificial diets consisting of crystalline cellulose (with and without lignin), α-cellulose (with and without lignin) and xylan. The termites were then dissected and the protist communities were analyzed through microscopy, leading to the conclusion that protist species are strongly influenced by diets. BIOLOG ECO Microplates® were used to assess the metabolic properties of the different types of consortia, highlighting strong differences on the basis of principal component analysis and calculation of similarity rates. The microorganisms were cultivated in liquid media corresponding to the artificial diets before being characterized through a metagenetic analysis of gut microbiota (16S ribosomal DNA). This analysis identified several phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Fibrobacteres, Firmicutes, Nitrospirae, OP9, Planctomycetes, Proteobacteria, Spirochaetes, TM6, Tenericutes, Verrucomicrobia and WS3. The OTUs were also determined and confirmed the abundance of Proteobacteria, Bacteroidetes, Firmicutes and Verrucomicrobia. It was possible to isolate several strains from the liquid media, and one bacterium and several fungi were found to produce interesting enzymatic activities. The bacterium Chryseobacterium sp. XAvLW produced α-amylase, β-glucosidase, endo-1,4-β-D-glucanase, endo-1,4-β-D-xylanase and filter paper-cellulase, while the fungi Sarocladium kiliense CTGxxyl and Trichoderma virens CTGxAviL generated the same activities added with endo-1,3-β-D-glucanase. [less ▲]

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical ... [more ▼]

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::GFP fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2D-gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein. [less ▲]

Zinc, lead and cadmium are metallic trace elements (MTEs) that are widespread in the environment and tend to accumulate in soils because of their low mobility and non-degradability. The purpose of this ... [more ▼]

Zinc, lead and cadmium are metallic trace elements (MTEs) that are widespread in the environment and tend to accumulate in soils because of their low mobility and non-degradability. The purpose of this work is to evaluate the applicability of biosensors as tools able to provide data about the bioavailability of such MTEs in contaminated soils. Here, we tested the genetically-engineered strain Escherichia coli pPZntAgfp as a biosensor applicable to the detection of zinc, lead and cadmium by the biosynthesis of green fluorescent protein (GFP) accumulating inside the cells. Flow cytometry was used to investigate the fluorescence induced by the MTEs. A curvilinear response to zinc between 0 and 25 mg/L and another curvilinear response to cadmium between 0 and 1.5 mg/L were highlighted in liquid media, while lead did not produce exploitable results. The response relating to a Zn2+/Cd2+ ratio of 10 was further investigated. In these conditions, E. coli pPZntAgfp responded to cadmium only. Several contaminated soils with a Zn2+/Cd2+ ratio of 10 were analyzed with the biosensor, and the metallic concentrations were also measured by atomic absorption spectroscopy. Our results showed that E. coli pPZntAgfp could be used as a monitoring tool for contaminated soils being processed. [less ▲]

BACKGROUND: Single cell biology has attracted a lot of attention these past few years and has led to numerous fundamental results pointing out the heterogeneity of clonal cell populations. In this context ... [more ▼]

BACKGROUND: Single cell biology has attracted a lot of attention these past few years and has led to numerous fundamental results pointing out the heterogeneity of clonal cell populations. In this context, microbial phenotypic heterogeneity under bioprocessing conditions needs to be further investigated. In this study, yeast based processes have been investigated by using on-line flow cytometry in combination with a fluorescent transcriptional reporter (GFP) and viability fluorescence tags (propidium iodide, PI). Methods aiming at expressing the dispersion of these fluorescence tags among the yeast populations have been investigated for different bioreactor operating conditions. RESULTS: Yeast viability was determined on the basis of PI uptake. Segregation between PI negative and positive subpopulations could be efficiently quantified on the basis of the mean-to-median ratio or the amplitude of the interquartile range. On the other hand, the same quantification could not be made for the segregation occurring at the level of GFP synthesis. Indeed, when cells were exposed to sub-lethal or mild stresses (such as in scale-down reactors) two GFP subpopulations could be visualized by real-time FC, but quantification by one of the above-mentioned methods was not possible. CONCLUSIONS: Yeast population heterogeneity was observed in representative bioreactor operating conditions. Difficulties for the determination of segregation at the level of GFP synthesis point out the fact that one needs to understand the segregation mechanisms for the applied fluorescent reporters, to judge whether simple mathematical tools may be applied or if more sophisticated computational tools are needed for the quantification of the microbial population segregation. [less ▲]

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas ... [more ▼]

A cellulolytic consortium was isolated from a composting plant in order to boost the initial hydrolysis step encountered in anaerobic digestion. Improvement of the cellulose degradation, as well as biogas production, was observed for the cultures inoculated with the exogenous consortium. Metagenomics analyses pointed out a weak richness (related to the number of OTUs) of the exogenous consortium induced by the selective pressure (cellulose as sole carbon source) met during the initial isolation steps. Main microbial strains determined were strictly anaerobic and belong to the Clostridia class. During cellulose anaerobic degradation, pH drop induced a strong modification of the microbial population. Despite the fact that richness and evenness were very weak, the exogenous consortium was able to adapt and to maintain the cellulolytic degradation potential. This important result point out the fact that simplified microbial communities could be used in order to increase the robustness of mixed cultures involved in environmental biotechnology. [less ▲]

Noise in gene and protein expression is a major cause for bioprocess deviation. However, this phenomenon has been only scarcely considered in real bioprocessing conditions. In this work, a scaling-law ... [more ▼]

Noise in gene and protein expression is a major cause for bioprocess deviation. However, this phenomenon has been only scarcely considered in real bioprocessing conditions. In this work, a scaling-law derived from a genome-scale studies based on GFP reporter systems has been calibrated to an on-line flow cytometry device, allowing thus to get an insight at the level of promoter activity and associated noise during a whole microbial culture carried out in bioreactor. We show that most of the GFP reporter systems investigated and thus corresponding genes could be included inside the area covered by the scaling-law. The experimental results suggest that this scaling-law could be used to predict the dynamics of promoter activity, as well as the associated noise, in bioprocessing conditions. The knowledge acquired throughout this work could be used for the design of more robust expression systems. [less ▲]

The application of sub-lethal stresses is known to be an efficient strategy to enhance survival of probiotic bacteria during drying processes. In this context, we previously showed that the application of ... [more ▼]

The application of sub-lethal stresses is known to be an efficient strategy to enhance survival of probiotic bacteria during drying processes. In this context, we previously showed that the application of heat stress upon the entry into stationary phase increased significantly the viability of Bifidobacterium bifidum. However, this heat shock has been considered only in small scale bioreactor and no information is available for a possible scaling-up strategy. Five different operating scales (0.2 L, 2 L, 20 L, 200 L and 2000 L) have thus been tested and the results showed that the viability of B. bifidum increases from 3.15 to 6.57 folds, depending on the scale considered. Our observations pointed out the fact that the heat stress procedure is scalable according to the main outcome, i.e. increases in cell viability, but other factors have to be taken into account. Among these factors, dissolved carbon dioxide seems to play a significant role since it explain the differences observed between the test performed at lab-scale and in industrial conditions. [less ▲]

In this work, the effect of iron oxide particles Fe2O3 and iron nanoparticles encapsulated in a porous silica matrix (xerogel Fe/SiO2) was investigated on biphenyl biodegradation by the strain Rhodococcus ... [more ▼]

In this work, the effect of iron oxide particles Fe2O3 and iron nanoparticles encapsulated in a porous silica matrix (xerogel Fe/SiO2) was investigated on biphenyl biodegradation by the strain Rhodococcus erythropolis T902.1. After 18 days of incubation biodegradation yields of 75% and 85% were achieved respectively in presence of non-autoclaved or autoclaved xerogel Fe/SiO2 at 10-5 M iron. These results are 42 and 60 % higher than in standard conditions without nanoparticles. They suggest that the autoclave procedure lead to the release of some iron less anchored in the silica matrix. This study highlights that siderophore production by Rhodococcus erythropolis T902.1 would be related to the presence of iron nanoparticles in the culture. It suggests that the production of these strong chelating compounds decreases with increase of iron release from xerogel Fe/SiO2. Moreover, most of the surfactants synthesized by Rhodococcus erythropolis T902.1 which are glycolipids containing trehalose (hexose), would be linked to cell surface and not excreted in the culture medium; the biomass hexose content also increased by 85% in presence of iron nanoparticles. [less ▲]

Steam explosion is a thermo-mechanicochemical pretreatment which allows the breakdown of lignocellulosic structural components by the action of heating, formation of organic acids during the process, and ... [more ▼]

Steam explosion is a thermo-mechanicochemical pretreatment which allows the breakdown of lignocellulosic structural components by the action of heating, formation of organic acids during the process, and shearing forces resulting in the expansion of the moisture. Two distinct stages compose the steam-explosion process: vapocracking and explosive decompression which include modification of the material components: hydrolysis of hemicellulosic components (mono- and oligosaccharides released), modification of the chemical structure of lignin, and modification of the cellulose crystallinity index, etc. These effects allow the opening of lignocellulosic structures and influence the enzymatic hydrolysis yield of the material. [less ▲]

Fermentative hydrogen production has often been described as inhibited by its own gas production. In this work, hydrogen production by Clostridium butyricum was investigated in batch Biochemical Hydrogen ... [more ▼]

Fermentative hydrogen production has often been described as inhibited by its own gas production. In this work, hydrogen production by Clostridium butyricum was investigated in batch Biochemical Hydrogen Potential (BHP) tests and in a 2.5L anaerobic sequenced batch reactor (AnSBR) under different operating conditions regarding liquid-to-gas mass transfer. Through the addition of both stirring up to 400rpm and nitrogen sparging, the yields were enhanced from 1.6 to 3.1molH2molglucose -1 and the maximum hydrogen production rates from 140 to 278mLh-1. These original results were achieved with a pure Clostridium strain. They showed that hydrogen production was improved by a higher liquid-to-gas hydrogen transfer resulting in a lower dissolved hydrogen concentration in the culture medium and therefore in a lower bacterial inhibition. In addition, biohydrogen partitioning between the gas and the liquid phase did not conform to Henry's Law due to critical supersaturation phenomena up to seven-fold higher than the equilibrium conditions. Therefore, dissolved hydrogen concentration should be systematically measured instead of the headspace hydrogen partial pressure. A model was proposed to correlate H2 production yield and rate by the pure C. butyricum strain CWBI1009 with mass transfer coefficient KLa. [less ▲]